P16INK4a expression is a surrogate marker of HPV E7-mediated pRB catabolism and is a sensitive and specific marker of high-grade premalignant and malignant lesions in cervical tissue biopsies. The utility of P16INK4a as a diagnostic adjunct for cervical cytology however, remains relatively unexplored. The goal of this R21/R33 phased innovation and application proposal is to evaluate the expression of P16INK4a as a diagnostic adjunct in cases with low-grade cytologic abnormalities including ASC-US, ASC-H, LSIL, and AGC, to identify cases that are most likely to have underlying high-grade premalignant lesions on cervical biopsy, The specific aims of the R21 application are: 1) to validate the immunocytochemical method for the detection of P16INK4a in cervical cytology specimens; 2) to define quantitative criteria for scoring P16INK4a test results; 3) to develop a pilot series of "gold standard" cytology specimens for P16INK4a analysis; and 4) to perform a preliminary evaluation of P16INK4a cytology test performance in "gold standard" cytology cases. The specific aims of the R33 proposal are: 1) to determine the correlation between the P16INK4a and high risk HPV detection with cytologic diagnoses in cases with normal cytology and in cases with ASC-US, ASC-H, LSIL, and AGC; 2) to determine the correlation between P16INK4a and high risk HPV detection in low grade cervical cytology specimens with the biopsy diagnosis of high-grade squamous or glandular lesions of the cervical squamous mucosa; 3) to determine the correlation between pl6lNK4a detection in low grade cervical cytology specimens with the histologic distribution of pl6lNK4a in concurrently collected cervical biopsies; and 4) to determine the optimal combination of cytology test procedures to maximize sensitivity and specificity for the detection of high-grade lesions on cervical biopsy. If successful, this study will provide a mechanism to identify patients that require aggressive clinical management and to defer the aggressive management of patients that will not benefit from further diagnostic evaluation.